| Research Backgrounds and Objective:Esophageal carcinoma, which threatens human being’s life and health,is one of the most common malignant cancers around the world, and thenumber of cases in China are accounting for over half of the global casesevery year, with esophageal squamous cell carcinoma (ESCC) the mostcommon. As one of the important treatments for esophageal carcinoma,radiation therapy is relatively poor in effects. The5-year survival rates areless than10%. Local recurrence and metastasis are the main causes forradiotherapy failure. However, the mechanisms of local recurrence andmetastasis after radiotherapy are still unclear.Epithelial-mesenchymal transition (EMT) refers to the phenomenonthat the epithelial cells lost their features and polarities while acquiring themesenchymal-like markers and invasive potential. EMT not only play key role in invasion and transference, but also have close relations with cancercells’ chemoresistance and cancer stem cells (CSCs) formation.Researches have showed that ionizing radiation (IR) could cause EMTand enhance the ability of transference in cancer cells. However, themechanisms of EMT induced by IR are not fully understood yet, and theinformation about whether EMT induced by IR is connected to cancer cells’acquired radioresistance has not been reported at home and abroad.This study means to explore the effects and mechanisms of acquiredtransference ability and radioresistance induced by IR-mediated EMT inESCC, finding out the key targets, with hopes of reducing the risk of localrecurrence and metastasis after radiotherapy through reversing EMT, andproviding experimental basis and theoretical foundation for improvingradiotherapy effect.Methods:1. Microscope was used to observe the morphology of esophagealcarcinoma cell lines Eca109, Te1and radiotherapy-resistant Eca109cells(Eca109R). Immunofluorescent test was used to detect the marker proteinsof EMT induced by IR in Eca109and Te1cells. Real Time PCR andWestern blot were used to detect the expression level of markers’ mRNAand proteins after EMT induced by IR in Eca109, Te1and Eca109R cells,respectively. Immunohistochemistry was used to test the expression ofEMT marker proteins in Eca109-maded nude mice xenografts after IR. Scratch test and transwell migration assay were used to detect the abilitiesof invasion and migreation in Eca109and Te1cells after IR. Colonyformation assay was used to test the radiosensitivity of Eca109andEca109R cells after5Gy irradiation.2. ELISA test was used to detect the level of IL-6secreted by Eca109and Te1cells after IR. Real Time PCR and Western blot were respectivelyused to detect the expression level of the mRNA and proteins related toIL-6/Stat3/Twist signaling pathway in Eca109and Te1cells after IR, aswell as the mRNA and protein expression level of Twist in Eca109R cells,which is a key factor in EMT transcription.3.30cases of the operation resection samples of ESCC before andafter neoadjuvant radiotherapy were collected, and immunohistochemistrywas used to test the expression level of EMT marker proteins and theproteins related to IL-6/Stat3/Twist signaling pathway of these samples.The difference in expression was then analyzed.4. AG490, special blockers of Jak2, and sh-Twist were used to blockthe IL-6/Stat3/Twist signaling pathway. Western blot was used to detect theexpression level of EMT marker proteins and IL-6/Stat3/Twist signalingpathway-related proteins in Eca109and Te1cells treated with IR, AG490and sh-Twist. Immunohistochemistry was used to test the expression levelof EMT marker proteins in Eca109-maded nude mice xenografts treatedwith IR, AG490and sh-Twist. Scratch test and transwell migration assay were used to detect the abilities of invasion and migration in Eca109andTe1cells treated with IR, AG490and sh-Twist.5. RNA interference (RNAi) was used to silence the expression ofTwist (a key factor in EMT transcription) in Ecal109R cells. Real TimePCR and Western blot were respectively used to detect the mRNA andproteins expression of EMT markers and Twist in Ecal109R cells. Colonyformation assay was used to test the radiosensitivity of Ecal109R cells.6. MTT assay was used to test the survival rate of Ecal109R cellstreated with IR and sh-Twist. Flow cytometry was used to detect theapoptosis. Establishing Eca109-maded transplantation tumor models ofnude mice and observing tumor growth. TUNEL test was used to detectapoptosis of exnografts. Western blot was used to detect the expressionlevel of Akt signaling pathway-related proteins and apoptosis-relatedproteins in Ecal109R cells.Results:1. The shape of Eca109, Te1cells and radiotherapy-resistant Eca109cells (Eca109R) after IR turned from round and ellipses into spindle.Compared with cells without radiotherapy, the expression of epithelialmarker E-cadherin was dramatically decreased in Eca109, Te1andEca109R cells after IR. However, the expression of mesenchymal markersN-cadherin and Vimentin were obviously increased. Compared with cellswithout radiotherapy, the expression of E-cadherin decreased in Eca109-maded xenografts after10Gy irradiation, with N-cadherin andVimentin increased dramatically. In addition, the abilities of invasion andmigration in Eca109, Te1cells were increased after IR, and theradioresistance was enhanced in Eca109and Eca109R cells after5Gyirradiation.2. There were significant differences of the expression of EMT markerproteins and the proteins related to IL-6/Stat3/Twist signaling pathwaybefore and after neoadjuvant radiotherapy. Compared with beforeradiotherapy, the rate of tissues with high E-cadherin expression wasdecreased after neoadjuvant radiotherapy, whereas the rates of tissues withhigh expression of N-cadherin, Vimentin and IL-6/Stat3/Twist signalingpathway proteins were increased.3. Compared with the mere radiotherapy group, the expression ofepithelial marker E-cadherin dramatically increased in Eca109, Te1cellstreated with AG490and sh-Twist. However, the expression ofmesenchymal markers N-cadherin an d Vimentin were obviously decreased,as well as p-Stat3and Twist. Compared with with the mere radiotherapygroup, E-cadherin expression increased dramatically in Eca109-madedxenografts treated with AG490and sh-Twist, whereas N-cadherin andVimentin expression decreased obviously. At the same time, the abilities ofinvasion and migration in Eca109, Te1cells were decreased after treatmentof AG490and sh-Twist. 4. Compared with the Eca109R/sh-NC group, the expression ofepithelial marker E-cadherin increased dramatically in Eca109R/sh-twistcells, whereas the expression of mesenchymal markers N-cadherin andVimentin obviously decreased. In addition, compared with Eca109R/sh-NCgroup, the cloning efficiency of Eca109R/sh-twist cells was decreased afterradiotherapy, with radiosensitivity increased.5. Compared with the Eca109R/sh-NC group, the Eca109R/sh-twistcells showed a decreased survival rate, increased apoptosis rate, slowergrowth rate and increased number of dead cells in xenografts afterradiotherapy.6. Compared with the Eca109R/sh-NC group, the expression of p-Aktand anti-apoptosis protein decreased in the Eca109R/sh-twist cells, withapoptosis protein expression increased.Conclusion:1. EMT induced by IR enhances migration ability and radioresistancein esophageal squamous cell carcinoma cells.2. IR promotes EMT through activating IL-6/Stat3/Twist signalingpathway in esophageal squamous cell carcinoma cells.3. Neoadjuvant radiotherapy promotes EMT through activatingIL-6/Stat3/Twist signaling pathway in esophageal squamous cell carcinomatissues.4. Blocking IL-6/Stat3/Twist signaling pathway leads to reversed EMT induced by IR and acquired metastatic potentiality in esophageal squamouscell carcinoma cells.5. Silencing Twist leads to reversed EMT and radioresistance ability inradiotherapy-resistant cells.6. Silencing Twist leads to increased effects of proliferation inhibitionand apoptosis promotion mediated by inactivated Akt. |
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